1. Field of the Invention
The present invention relates to means of generating blue laser light, and more specifically, it relates to the excitation of biofluorescent dyes (in the .about.490-496 nm spectral region) utilized in flow cytometry, immunoassay, DNA sequencing, and other biofluorescence instruments.
2. Description of Related Art
Fluorescent probe techniques are widely utilized in a variety of biomedical research and diagnostic applications, owing largely to inherently high detection sensitivity and selectivity. Since the early 1950s, fluorescent techniques have been used to probe molecular interactions between small ligands and biological macromolecules, as well as interactions among components in biological assemblies (e.g., cells). Applications soon extended to the areas of nucleic add and cell membrane research. Fluorescent probes have increasingly been used by cell biologists to probe cellular and subcellular function structures, as in fluorescence microscopy imaging and immunofluorescence microscopy techniques.
A flow cytometer [1] is often used to implement biofluorescence techniques. In a flow cytometer, cells in a fluid suspension are stained with fluorescent dyes that bind to specific molecules on the surface of the cell or to the nucleic acids within the cell nucleus. These cells then pass in single file through a focused laser beam which excites the fluorescent dye molecules. The wavelength of the laser is chosen to match the wavelength of a fluorescence excitation band of the dye molecule. Appropriately placed detectors capture a portion of the scattered and fluorescent light emitted by the cell as it passes through the laser beam. Several different fluorescent dyes may be employed, each emitting at a wavelength differing from that of the others. A corresponding number of appropriately filtered photomultiplier detectors measure the fluorescent intensities from their corresponding dye emitters. Each data stream represents a distinct measurement of information about immunological marker molecules on the cell surface or in the nucleic acids of the nucleus. The use of antibodies for cell identification is possible because the external membranes of different kinds of cells express different moieties called cell surface antigens. Immunotyping is used to describe the science of identifying cell using antibodies. For example, antibodies are commonly used to identify leukocytes in human blood. The development of multiparameter flow cytometry [1] for immunotyping clinical material has provided new insight on diagnosis and management of disease. Using this approach, considerable information can be rapidly obtained for the treatment of immuno-deficiency diseases, such as AIDs, to evaluate immunosuppressive therapy associated with organ transplantation, and to manage cancer therapy. Clinical immunotyping is mostly applied to cells from the blood or bone marrow using antibodies labeled with fluoscein and/or phycoerthrin dyes, excited in an excitation band peaked at a wavelength near 493 nm. Using different antibodies labeled with fluorescein and other fluorochromes such as phycoerthrin, multiple cell types can be identified simultaneously using a single cell suspension in a flow cytometer.
In the past decade there has been a precipitous increase in interest in, and the prospects for, genetic medicine based on a complete mapping of the human genome. To sequence the human genome [2], chromosomal DNA fragments are selectively stained chemically with one of four different fluorescent dyes (one fluorescent color for each of the four genetic letters A, G, T, C), and passed through a multi-channel electrophoresis sequencing apparatus). The multiple channels are illuminated with laser radiation matching the excitation bands of the fluorescent dyes, and the genetic letter sequences are readout by detecting the positions of the four fluorescence colors. Four color fluorescence DNA sequencing [2] has enabled the onset of an international campaign to sequence the complete human genome in a few years time.
In spite of the vast number of fluorescent probes that are available for research purposes, only a handful of fluorescent dyes have gained wide-spread use in commercial biofluorescence instrumentation (such as the Model 373A DNA Sequencer produced commercially by Applied Biosystems Inc. (ABI), Foster City, Calif.). The dyes developed specifically for wide-spread commercial use in most flow cytometers [1], immunoassay [3] and four color fluorescence DNA sequencers [4] were selected (in part) for their ability to be efficiently excited by the 488 nm or 514 nm radiation from an argon ion gas laser (the only visible laser source then deemed practical for use in commercial bio-fluorescence instruments. These standard biofluorescence dyes, and their excitation (fluorescence) maxima in nm, are as follows [8]: FITC (fluorescein isothiocyanate) 494 (518), NBD-HA (aminohexanoic acid) 492 (548), tetramethylrhodamine 548 (578), and Texas Red 580 (604). The ABI dye primer product names are FAM, JOE, TAMRA, and ROX. Another widely used commercial dye in flow cytometers for immunotyping is phycoerythrin. Although the 488 nm laser line of the argon ion laser is a few nm to the blue side of the FAM excitation peak, the argon ion laser as been found (until now) to be the best practical source for most commercial biofluorescence instruments utilizing FAM and other dyes, such as phycoerythrin. Typical laser excitation powers of a few milliwatts to a few tens of milliwatts are employed in commercial biofluorescence instruments.
New four color dye staining techniques have been recently developed [9] to increase DNA sequencing throughput and accuracy. The dye (FAM), with an excitation peak wavelength near 494 nm, is used to absorb 488 nm excitation radiation from an argon ion laser, and the excitation energy is transferred (nonradiatively) to energy transfer primers (designated F10F, F10J, F3T, and F3R) that fluoresce at peak wavelengths wavelengths of 525, 555, 580, 605 nm, respectively.
As biofluorescent research, diagnostic, and clinical instrumentation applications expand and proliferate, there is a pressing need to provide a more compact, efficient, reliable, and long-lived dye excitation laser source than the argon ion laser, for use in these instruments, especially in clinically used flow cytometers and in DNA sequencers. A large preponderance of these instruments utilize the fluorescein dye primer FAM that is excited optimumly in the blue spectral region peaked at .about.493 nm [8], or the fluorochrome phycoerythrin (peaked at the same wavelength).
Two leading approaches to producing generally blue laser sources are by (i) harmonic doubling of near infrared radiation from either AlGaAs (.about.860 nm) or InGaAs (.about.980 nm) laser diodes in quasi-phase-matched nonlinear optical waveguides [11], producing generally blue radiation near .about.430 nm and .about.490 nm, respectively; and (ii) harmonic doubling in bulk nonlinear optical crystals (such as KNbO3) of the near infrared radiation emitted from a diode-pumped, neodymium-doped solid state crystal laser operating on the quasi-three-level laser transition [12].
Direct harmonic doubling of AlGaAs laser diode radiation at .about.860 nm in quasi-phase-matched waveguides is the baseline approach of two national consortia devoted to developing short-wavelength (.about.430 nm) blue laser sources for the low power optical data storage application. This approach requires the use of relatively expensive frequency-stabilized laser diodes, matched to the conversion peak of the nonlinear doubling waveguide, and very close control of operating temperature [11]. Operating lifetimes of the nonlinear quasi-phased matched waveguides have proved too limited for commercial use [13]. Blue output powers have been generally limited to a few to tens of milliwatts, and prospects for the practical scaling of blue output power beyond tens of milliwatts is problematic.
The practical prospects of providing &gt;tens of milliwatts of generally blue light by harmonic doubling of the fundamental infrared radiation from a diode-pumped rare-earth-doped solid state laser emitting in the near infrared spectral region are relatively greater than the approach just described. However, as discussed below, the use of the trivalent neodymium (Nd) rare-earth-ion as the active laser ion cannot produce the desired color of blue radiation preferred for exciting commercial biofluorescent probe dyes, especially the FAM and phycoerythrin dyes near .about.493 nm.
To overcome the deficiencies of these two approaches, it is proposed to construct blue-radiation emitting diode-pumped solid state lasers (DPSSLS) using ytterbium-doped dielectric laser crystals, in particular preferred Yb doped apatite and tungstate crystals, operating on their resonance (true 3-level) ZPL transitions generally falling within in the spectral region from .about.965-992 nm. The generated fundamental radiation at a wavelength within the .about.965-992 nm spectral region is then harmonically doubled to the blue spectral region near .about.493 nm using a suitable nonlinear harmonic doubler.